Project Summary The goal of this research project is to design and apply fluorescence decay kinetic-based flow cytometry on a microchip platform. The system will be used to quantify Frster resonance energy transfer (FRET) events inside of mammalian cells and fully enrich near-infrared fluorescent proteins based on their photo-kinetics. The microflow device will incorporate unique features such as acoustic focusing of cells through microfluidic channels, multi-frequency measurements that give rise to multiple-fluorescence lifetime values per cell, imaging capabilities to capture multi-pixel fluorescence lifetime measurements, and sorting capabilities dependent on decay-kinetic parameters. Our first aim will be to use the cytometer to count cells based on changes in the fluorescence (FRET) donor?s changing fluorescence lifetime. When FRET is evaluated by the excited state kinetic changes of the energy-transferring fluorophore pairs, the result is a data set that has not been affected by intensity-based artifacts. Moreover, with new computational toolboxes including phasor-based FRET trajectories and FRET efficiency, cytometric parameters are developed for cell screening that provide heterogeneity of lifetimes within the cell at a rate of thousands of cells per second. We test this with FRET at the cell surface as well as with an intracellular FRET bioprobe. Both systems have biomedical significance related to protein function alteration thereof with targets during screening. The second aim for this project is to take the microchip-based system and use it to actively screen bacterial libraries and sort single cells that express near-infrared fluorescent proteins with high quantum yield. The quantum yield is a photophysical trait of fluorescent molecules that is directly proportional to the average fluorescence lifetime, or average time the fluorophore spends in the excited state. Therefore a tool that can isolate samples based on the fluorescence lifetime is quite valuable since the average intensity can be plagued by other factors such as concentration, quantum efficiency, and instrument artifacts. The long term significance of our second aim is the ability to expedite the development of near-infrared fluorescent proteins for use in molecular and diffuse optical tomography. In general, the development of a compact, sensitive, and time-dependent cytometry system is impacting beyond the two biomedical applications proposed. Accordingly this work is the first step toward evaluating the benefits, demonstrating the quantitative nature, and setting the stage for broad use across many more cytometric applications.